7406
Biochemistry 2005, 44, 7406-7413
FTIR Studies of Internal Water Molecules in the Schiff Base Region of Bacteriorhodopsin† Mikihiro Shibata and Hideki Kandori* Department of Materials Science and Engineering, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan ReceiVed January 21, 2005; ReVised Manuscript ReceiVed March 18, 2005
ABSTRACT: In a light-driven proton pump protein, bacteriorhodopsin (BR), three water molecules participate in a pentagonal cluster that stabilizes an electric quadrupole buried inside the protein. Previously, lowtemperature Fourier-transform infrared (FTIR) difference spectra between BR and the K photointermediate in D2O revealed six O-D stretches of water in BR at 2690, 2636, 2599, 2323, 2292, and 2171 cm-1, while five water bands were observed at 2684, 2675, 2662, 2359, and 2265 cm-1 for the K intermediate. The frequencies are widely distributed over the possible range of stretching vibrations of water, and water molecules at 80 cm-1 in T89A, but not in Y185F, strongly supports the proposal by the QM/MM calculation that the lowest O-D band originates from the O-D stretch of water402 interacting with Asp85, though the frequency from the experiment (2171 cm-1) is considerably different from that from the calculations (2257 cm-1) (12). From where do other vibrations originate? Previous QM/ MM calculation predicted that the second lowest O-D stretch is water401 interacting with Asp85 [2320 cm-1 (Table 1)], and the third lowest O-D stretch is water406 interacting with Asp212 [2468 cm-1 (Table 1)], while the O-D stretch of water402 interacting with Asp212 is at a higher frequency [2618 cm-1 (Table 1) (13)]. We further attempted to identify these water vibrations experimentally by use of mutants at the extracellular side. Figure 5 shows the K minus BR difference spectra of the wild type and mutant proteins of BR at the extracellular side in the 2380-2000 cm-1 region. The 2292 (-)/2265 (+) cm-1 bands disappear in R82Q (Figure 5b), whereas the water bands at 2359 (+), 2323 (-), and 2171 (-) cm-1 are preserved with small changes in their frequencies. This observation strongly suggests that the water bands at 2292 and 2265 cm-1 are highly influenced by the mutation at position 82. The most straightforward interpretation is that the water bands at 2292 and 2265 cm-1 originate from water406 interacting with Asp212 in BR and K, respectively. As described above, the 2292 (-)/2265 (+)
7410 Biochemistry, Vol. 44, No. 20, 2005
FIGURE 5: K minus BR difference infrared spectra of wild type (a), R82Q (b), R82Q/D212N (c), and E204Q (d) BR in the 23802000 cm-1 region. The samples were hydrated with D2O (red lines) or D218O (blue lines), and spectra were measured at 77 K. One division of the y axis corresponds to 0.002 absorbance unit. Labeled frequencies correspond to the bands identified as water stretching vibrations.
cm-1 bands also contain the N-D stretch of Arg82 (16). Removal of the hydrogen bond between Arg82 and water406 is likely to weaken the hydrogen bond of water406 interacting with Asp212. The 2292 (-)/2265 (+) cm-1 bands disappear in R82Q, whereas the 2359 (+)/2323 (-) cm-1 bands of the wild type are likely to appear at 2356 (+)/2323 (-) cm-1. The water O-D stretch at 2323 cm-1 may originate from (i) water401 interacting with Asp85, or (ii) water402 interacting with Asp212, postulating that this water hydrates a negative charge. According to the QM/MM calculation, however, the latter is under weak hydrogen-bonding conditions [2618 cm-1 (Table 1)], while the O-D stretch of water401 is at 2320 cm-1, being in good agreement with our FTIR observations (13). In fact, Figure 6c shows the presence of the negative 2339 cm-1 band in R82Q/D212N, suggesting that the water possessing the O-D stretch at 2323 cm-1 does not form a hydrogen bond with Asp212. In E204Q, water bands were observed 2363 (+), 2292 (-), 2280 (+), 2244 (-), and 2171 (-) cm-1 (Figure 5d). The water bands at 2292 (-)/2280 (+) cm-1 are likely to be the same as those at 2292 (-)/2265 (+) cm-1 for the wild type (Figure 5a), because they are highly dichroic like those of the wild type (data not shown). Thus, among strongly hydrogen-bonded water bands, it seems that only the band at 2323 cm-1 in the wild type is downshifted by ∼80 cm-1 to 2244 cm-1 in E204Q. Glu204 is distant from the Schiff
Shibata and Kandori
FIGURE 6: K minus BR difference infrared spectra of wild type (a), D85N (b and c), D212N (d), Y185F (e), and T89A (f) BR in the 2720-2570 cm-1 region. The samples were hydrated with D2O (red lines) or D218O (blue lines), and spectra were measured at 77 K, except for spectrum c. One division of the y axis corresponds to 0.002 absorbance unit. Labeled frequencies correspond to the bands identified as water stretching vibrations.
base region, whereas it is known that the pKa of Asp85 is increased in E204Q, indicating the presence of a longdistance hydrogen-bonding network (17, 18). The strengthened hydrogen bond of water401 with Asp85 in E204Q is possibly correlated with the higher pKa value of Asp85. On the basis of the results described above, we concluded that the water bands at 2171, 2292, and 2323 cm-1 originate from the O-D stretches of water402, -406, and -401, respectively. From the previous results in H2O (9), we also estimated the corresponding O-H stretches to be at ∼2800, ∼3000, and 3100 cm-1 for water402, -406, and -401, respectively (Table 1). It should be noted that a water molecule generally possesses coupled stretching vibrations, symmetric and antisymmetric modes, at nearby frequencies (1). The absence of the other vibrations in this frequency region strongly suggests that vibrational coupling is small for the water molecules in the pentagonal cluster. It would be reasonable because each water molecule interacts with a negative charge. We expect the other stretching vibrations to appear in a higher-frequency region. Identification of the Water O-D Stretches under Weak Hydrogen- Bonding Conditions. Figure 6a shows the K minus BR difference spectrum of the wild type in the 2720-2570 cm-1 region. There are three water bands for both positive and negative sides. The previous QM/MM calculations
Schiff Base Waters in Bacteriorhodopsin
Biochemistry, Vol. 44, No. 20, 2005 7411
FIGURE 8: Schematic drawing of the deuterated pentagonal cluster structure in the Schiff base region. The numbers are the O-D stretching frequencies of water molecules in inverse centimeters assigned in this FTIR study.
FIGURE 7: K minus BR difference infrared spectra of wild type (a), R82Q (b), R82Q/D212N (c), and E204Q (d) BR in the 27202570 cm-1 region. The samples were hydrated with D2O (red lines) or D218O (blue lines), and spectra were measured at 77 K. One division of the y axis corresponds to 0.002 absorbance unit. Labeled frequencies correspond to the bands identified as water stretching vibrations.
reproduced the O-D stretches of water molecules in BR rather well (Table 1) (13). Here we examine the origin of water bands by use of mutant proteins. In the case of D85N, only one negative water band (2599 cm-1) was observed at 77 K (Figure 6b), which is in high contrast to the wild type. However, the spectra of D85N at 130 K exhibited the presence of three water bands at the negative side (Figure 6c). We found that the mutants of Asp85, such as D85N, D85S, and D85T, exhibit temperaturedependent water bands between 77 and 130 K, whereas other mutants were not influenced by such temperature changes (data not shown). Water bands of D85N were significantly different from those of the wild type even at 130 K. The spectra of D212N possess water bands at 2684 (-), 2675 (+), 2652 (-), 2631 (+), 2616 (-), and 2601 (+) cm-1 (Figure 6d), which are different from those of the wild type as well. It must be noted that D85N and D212N entirely lack water bands under strong hydrogen-bonding conditions (spectra b and c of Figure 4, respectively), which might indicate the absence of water molecules in the pentagonal cluster. However, the presence of three negative bands of water for D85N and D212N (spectra c and d of Figure 6, respectively) suggests that these mutants also contain internal water molecules in the Schiff base region like the wild type, whereas the hydrogen-bonding interaction of such water molecules is different. Spectra e and f of Figure 6 show the difference FTIR spectra for Y185F and T89A, respectively. The spectra of
Y185F possess water bands at 2690 (-), 2684 (+), 2675 (+), 2655 (+), 2624 (-), and 2599 (-) cm-1, whose spectral features are similar to those of D212N (Figure 6d). In contrast, the spectra of T89A possess water bands at only 2649 (-) and 2618 (+) cm-1, whose spectral features are similar to those of D85N (spectra b and c of Figure 6, respectively). These facts suggest the importance of the hydrogen-bonding interactions between Asp85 and Thr89 and between Asp212 and Tyr185. Figure 7 shows the K minus BR difference spectrum of the wild type and the mutants of amino acids at the extracellular side in the 2720-2570 cm-1 region. R82Q possesses water bands at 2686 (-), 2673 (+), 2646 (+), and 2590 (-) cm-1 (Figure 7b). Additional mutation at position 212 significantly alters the spectral shape, exhibiting water bands at 2690 (+), 2684 (-), 2649 (-), and 2625 (+) cm-1 (Figure 7c). In the case of E204Q, the negative water band of the wild type at 2690 cm-1 seems to disappear, while there are three positive bands at 2684, 2675, and 2662 cm-1 together with a negative band at 2636 cm-1. From these FTIR spectra of various mutants, it is likely that three water O-D stretches at 2690, 2636, and 2599 cm-1 originate from water401, -402, and -406, respectively, in the pentagonal cluster (Figure 8). Because of secondary effects of mutations, however, it is difficult to determine the origin of the water O-D stretches, particularly for weakly hydrogenbonded water molecules. The model of the pentagonal cluster in BR predicts that one O-D stretch of water401 is free of hydrogen bonds (Figure 8). Therefore, it is a reasonable postulation that this O-D stretch has the highest frequency, namely, at 2690 cm-1 (Figures 6 and 7). In fact, the O-D stretch at 2690 cm-1 in D2O corresponds to the O-H stretch at 3643 cm-1 in H2O, which is almost free of hydrogen bonds. Such assignment is supported by the fact that the 2690 cm-1 band is not significantly affected by the mutations at Asp212 and Tyr185 (spectra d and e of Figure 6), while disappearing in D85N (77 K, Figure 6b), T89A (Figure 6f), and E204Q (Figure 7d). Thus, we conclude that the water O-D stretch at 2690 cm-1 originates from water401 that is
7412 Biochemistry, Vol. 44, No. 20, 2005 free of hydrogen bonds. In contrast, we could not assign the water bands at 2636 and 2599 cm-1 uniquely. We follow the assignment of the QM/MM calculations (13), and eventually six O-D stretches in BR were identified as shown in Table 1 and Figure 8. DISCUSSION In this article, we attempted to identify the origin of the water O-D stretches observed in the low-temperatrue FTIR difference spectra of BR at 77 K. To accomplish that goal, we extensively studied the K minus BR difference spectra of various mutant proteins, with mutations not only in the Schiff base region but also in the cytoplasmic and extracellular regions. We found that the water bands were not affected by the mutations at the cytoplasmic side, but significantly altered by the mutations in the Schiff base region and at the extracellular side. We concluded that the six O-D stretches of BR originate from three water molecules, water401, -402, and -406, in the pentagonal cluster (Figure 1b), which is consistent with the previous QM/MM calculations (13). It should be noted that the waters in the pentagonal cluster are not necessarily close to the retinal chromophore. The distances from water401, -402, and -406 to the nearest atom of the chromophore, which is the Schiff base nitrogen, are 6.1, 2.9, and 5.9 Å, respectively (6). Water501, -502, and -511 are 4.4 Å from the methyl carbon at C13, 7.7 Å from C15, and 9.4 Å from C10, respectively (Figure 1a). Water407, -403, and -405 are 9.6 Å from the Schiff base nitrogen, 9.8 Å from C8, and 8.8 Å from the methyl carbon at C2, respectively (Figure 1a). These facts suggest the importance of the hydrogen-bonding network, where watercontaining pentagonal cluster structure must play a crucial role. While the water bands in the K minus BR spectra were not affected in the mutants at the cytoplasmic side such as T46V, D96N, and D115N (Figures 2 and 3), significant modifications of the water bands were observed for the mutations at the extracellular side such as R82Q, R82Q/ D212N, and E204Q (Figures 5 and 7). It is well-known that the pKa values of Asp85 and the proton release group are correlated with each other, because the mutations of Arg82 and Glu204 increase the pKa of Asp85 (17-19). Thus, the importance of an extended hydrogen-bonding network has been discussed. Our FTIR study observed that the 2171 and 2323 cm-1 bands downshift to 2159 and 2244 cm-1 for R82Q and E204Q, respectively (Figure 5). Since they can be ascribed to the water O-D stretches interacting with Asp85 (Figure 8), the stronger hydrogen bond of water (manifested by the shift to a lower frequency) is presumably correlated with the change in the pKa of Asp85. Figure 8 summarizes the O-D stretches of the water molecules in the pentagonal cluster. Two stretching modes of these waters are highly separate in frequency (300-470 cm-1 for O-D stretches and 500-770 cm-1 for O-H stretches) (Table 1). In general, a water molecule has two O-H groups, and their frequencies are distributed in the wide 3700-2700 cm-1 region dependent on their coupling and hydrogen-bonding strength. Gaseous water exhibits asymmetric and symmetric stretching modes at 3755 and 3657 cm-1, respectively, and the stretching frequency is lowered
Shibata and Kandori as its hydrogen bond becomes stronger (20). It must be noted that the hydrogen-bonding strengths of the two O-H groups are probably not equivalent in the restricted protein environment, which breaks the C2V-type symmetry. In such CS-type symmetry, one O-H group is hydrogen-bonded and the other O-H group is unbonded, and their frequencies are widely split (1). These results can be explained well by such decoupling of the two stretching modes. Small vibrational coupling is presumably due to strong association of the waters with negative charges at Asp85 and Asp212. The frequencies of water402 are particularly prominent. As we reported previously, the 2171 cm-1 band was assigned as the O-D stretch of water402 hydrating Asp85 (12). This FTIR study together with the QM/MM calculations strongly suggests that the other O-D stretch of water402 is at 2636 cm-1 (or 2599 cm-1). Therefore, the frequency difference between the two O-D stretches is as large as 465 cm-1, which corresponds to ∼770 cm-1 for O-H stretches (Table 1). In the unphotolyzed state of BR, the position of water402 looks symmetrical with respect to Asp85 and Asp212 (Figure 1b). This study, however, showed highly asymmetric association of water402 with the aspartates. Since the hydrogenbonding strength of water is closely related to the geometry of the O-H (O-D) group, such asymmetric interaction of water402 presumably originates from the orientation of their O-H (O-D) groups. As a consequence, water402 bridges the Schiff base nitrogen and an oxygen of Asp85 ideally. Formation of the K intermediate is accompanied by hydrogen-bonding alterations of the water molecules in the pentagonal cluster. This FTIR study of R82Q (Figure 5b) suggests that the 2292 cm-1 band of water406 in BR corresponds to the 2265 cm-1 band in K, which also overlaps with the N-D stretch of Arg82 (16). According to the QM/ MM calculations by Hayashi et al., the 2690 and 2323 cm-1 bands of water401 correspond to the 2684 and 2359 cm-1 bands in K, respectively (21). Although the QM/MM calculations for the K intermediate explain this FTIR observation well, one exception is the O-D stretch of water402 hydrating Asp85. The QM/MM calculations resulted in the O-D stretches at 2257 and 2423 cm-1 for BR and K, respectively (21). The observed O-D stretch in BR is at 2171 cm-1 (Figure 8), whereas the corresponding O-D stretch in K is not determined in this study. There are no positive water bands around 2423 cm-1 in the K minus BR spectra (10, 11). We observed only five positive bands for K (Figures 2 and 3). It is possible that the positive band is present in the 2300-2200 cm-1 region, where it clearly cannot be observed. Further experimental and theoretical efforts are necessary to fully understand the water bands in the K intermediate. Among various mutant proteins we studied here, only D85N and D212N lack strongly hydrogen-bonded water molecules (
